The amyloid- peptide (A) and the microtubule-associated protein tau are both strongly implicated in the pathogenesis of Alzheimer's disease (AD). However, the role of tau in AD pathogenesis is not well understood, and tau is underdeveloped as a therapeutic target. Major clues to its pathogenic role are nearly 40 mutations in the tau gene that cause certain forms of frontotemporal dementia (FTD), a tauopathy related to AD. Many of these mutations are silent and alter the splicing of the tau pre-mRNA, leading to increased inclusion of exon 10 and thereby shifting the balance between tau proteins that contain 3 or 4 microtubule binding domains (3R and 4R tau, respectively). We have validated a postulated stem-loop at the boundary between exon 10 and intron 10 as a bona fide structure that regulates 3R versus 4R tau formation in cells. We have also carried out a high-throughput screen to identify small drug-like compounds that bind to and stabilize this stem-loop structure as a potential therapeutic strategy, determined the structure of one of these compounds bound to the tau mRNA stem-loop by NMR, and validated this structural model through analogue design. In addition, we have developed novel antisense molecules that recognize the discontinuous mRNA sequences that flank the tau pre-mRNA stem-loop structure and skew tau pre-mRNA splicing away from the 4R isoforms. These antisense molecules are bipartite, connected via a linker region, with one part complementary to the sequence just upstream from the stem-loop and the other complementary to the sequence immediately downstream. Such bipartite antisense molecules could be said to be both sequence- and structure-specific, as simultaneous binding to the two flanking regions requires stem-loop formation. In light of these findings, we now propose to combine these two approaches and develop antisense-MTX conjugates that stabilize the tau stem-loop structure with high potency and selectivity. Because MTX binds near the bottom of the stem, close to the sites where antisense molecules bind, linking MTX to such antisense molecules is expected to result in highly potent and specific molecular clasps that simultaneously target the tau pre-mRNA stem-loop structure, sequences upstream of this structure, and sequences downstream of this structure. The result of such tripartite binding by these molecular clasps would be potent and selective alteration of tau splicing in a therapeutically beneficial way. Specifically, we propose o: (1) generate antisense-MTX conjugates, (2) test these conjugates for their ability to bind to and stabilize the tau pre-mRNA stem-loop structure, and (3) determine the ability of these conjugates to shift splicing of the tau pre-mRNA away from the 4R isoforms. PUBLIC HEALTH RELEVANCE: Targeting Tau Splicing for Dementia RNA is a critical intermediate in the pathway from DNA (genes) to their encoded proteins. These RNA intermediates undergo complex alternative processing that ultimately leads to variations of the proteins. Such complex processing occurs with an important RNA molecule involved in Alzheimer's disease and related dementias, an RNA encoding a protein called tau. In this project, we aim to develop a strategy for controlling the alternative processing of the tau RNA with small drug-like molecules linked to larger polymeric molecules, agents we have dubbed molecular clasps.